Using a vaccinia virus recombinant system, we demonstrated before that self-assembly of p55 GAG protein is sufficient to form the skeletal framework of the nascent human immunodeficiency virus (HIV-1) particle. The particles which budded from the cells infected with a vaccinia-GAG construct were mostly spheres with a concentric ring of electron dense material. Expression of the GAG and POL proteins resulted in mature particles containing a condensed core which assumed the nucleoid structure characteristic of lentiviruses. When the POL frame in the GAG-POL ORF was truncated at the end of the protease domain, p55 GAG processing was markedly reduced and the maturation of the resultant particles was defective. The mechanism of RNA encapsidation during retrovirus particle formation is still unclear. In particular, the nature and the identity of the RNA sequences and the gag subunits involved in HIV capsidation remain to be solved. We have now developed a simple in vivo model for HIV-1 RNA packaging. A subgenomic HIV RAN encompassing R, U5 and U3 elements surrounding the minimal packaging sequence was expressed from the T7 promoter using vaccinia T7 polymerase vector. Co-infection with GAG or GAG-POL recombinant vaccinia viruses led to HIV particles that selectively packaged the mini HIV RNA. We have expressed cleavage defective mutants of GAG using the vaccinia vector. Mutations that eliminate the myristoylation or the nucleic acid binding CYS containing motif (NBCys) of the p9 subunit of GAG protein have been analyzed in the context of GAG-POL expression to examine whether they interfere with RNA packaging. This system is being used to define the molecular parameters of RNA capsidation. It is our goal eventually to develop molecular strategies to interfere with RNA packaging and particle maturation.